In a clinical setting, urine specimens may contain significant numbers of contaminating blood cells (17, 21). To demonstrate peptide selectivity for the urothelial cells of interest, moderate to heavy contamination was simulated by applying mixtures of WBCs and J82 cells (start ratio 7:1, 8:1, or 72:1) to the slides. The end ratio of WBC:J82 cells captured on the slides was calculated after washing away unbound cells. The observed WBC depletion was ≥81% for all peptide-conjugated slides, with depletion ≥95% for 3 of the peptides (Table 2). Meanwhile, sensitivity of urothelial cell capture was measured by applying low numbers of J82 cells (48–384 cells) to individual peptide-conjugated wells. The percent recovery increased with increasing input cell number, ranging 40%–75% within the clinically relevant range (Table 3).Table 2.
To demonstrate that the peptide-conjugated slides enrich for urothelial cells from urine contaminated with whole blood, J82 cells and anti-coagulated whole blood were spiked into fresh urine. The cells were deposited on slides, fixed, and stained with H&E (Figure 1). Samples prepared on poly- L-lysine slides were covered predominantly with red blood cells. Attempts to wash away the obscuring blood cells prior to fixing also removed the J82 cells, rendering the slides inadequate for subsequent analysis. However, peptide-conjugated slides could be rinsed to wash away the RBCs and WBCs, leaving the J82 cells on the slide for evaluation. Thus, the peptide-conjugated slides selectively capture scarce urothelial cells out of mixed populations heavily dominated by obscuring blood cells, making them superior substrates for diagnostic sample preparations from patients presenting with gross hematuria.
Capture of endogenous urothelial cells
Endogenous cells were obtained by processing voided urine obtained from a healthy donor. Recovered cells were applied to peptide-conjugated slides and subjected to Papanicolaou staining. Small basal urothelial cells as well as larger intermediate and superficial cells were observed with clearly distinguished nuclei (Figure 2). To assess peptide specificity for capture of endogenous urine cells, slides were prepared with four distinct regions. While unmodified regions captured only 8 cells from 40 mL of voided urine, the peptide-conjugated regions captured 27–59 cells (Table 4). When comparing the types of cells captured, EBP-8 and EBP-35 captured 3.5-fold and 2.1-fold more diagnostic basal and intermediate cells, respectively, while EBP-37 captured 7-fold more superficial and squamous cells. In a research or clinical setting, slides with multiple peptide-conjugated spots would thus enable investigators to capture, purify, and analyze multiple cell types on one slide using a single processing step. Together, these experiments demonstrate that the peptide-conjugated slides maintain their specificity for normal and cancerous urothelial cells when processing human urine specimens. Furthermore, the slides are compatible with Papanicolaou staining, the most prevalent method for evaluating cells on clinical cytology slide preparations (22).
Characterization of captured urothelial cells
In the hands of inexperienced cytopathologists, cytology can have a lower sensitivity than FISH (23). Meanwhile, ICC can have a higher sensitivity than cytology for low-grade cancers (24, 25). As a result, the more expensive ICC- and FISH-based tests are commonly used to aid in the evaluation of epithelial cells deposited on slides. Carcinoembryonic antigen (CEA) is a membrane-associated cancer marker used in the FDA-approved ImmunoCyt ICC test to monitor bladder cancer patients, while survivin is a nucleus- and/or cytoplasm-associated cancer marker expressed in transitional bladder carcinoma cells that is correlated with poor clinical outcomes (26). Detection of CEA and survivin in cancer tissue and circulating tumor cells has prognostic and predictive relevance and may serve to guide treatment options.
To confirm that peptide binding does not interfere with ICC analysis, we evaluated the expression of CEA and survivin on peptide-conjugated slides. J82 urothelial cells were captured on peptide-conjugated slides, and their CEA expression was compared with cells deposited on positively charged slides or poly-L-lysine slides, two slide types commonly used for cytology preparations and ICC (Figure 3). CEA expression area per cell on the positively charged and poly-L-lysine slides was 207.7 and 333.8 µm2, respectively. The average CEA expression area per cell on the peptide-conjugated slides fell within the same range (253.3–276.5 µm2) (Table 5). Meanwhile, sur vivin expression detected by ICC in the nuclei of captured cancer cells was similar on both the peptide-conjugated and poly- L-lysine slides (Figure 4). Together, these results indicate that the peptides do not interfere with slide-based ICC analysis. Finally, as most urine specimens are fixed prior to analysis, J82 cells were fixed in PreservCyt (Hologic, Bedford, MA) prior to capture on the slide. PreservCyt is the same fixative used with the ThinPrep system, and the capture and CEA marker expression of preserved cells on the peptide-conjugated slides was comparable to fresh cells (Figure 3E). Similarly, endogenous cells from urine fixed in PreservCyt were captured on peptide-conjugated slides in numbers comparable to fresh cells (data not shown).